Minimization of heavy metal toxicity in radish (Raphanus sativus) by strigolactone and biochar

Due to the high solubility of Cd in water, it is considered a potential toxin which can cause cancer in humans. In plants, it is associated with the development of oxidative stress due to the generation of reactive oxygen species. To overcome this issue, the roles of different plant hormones are vital. Strigolactones, one of such natural plant hormones, show promise in alleviating cadmium toxicity by mitigating its harmful effects. Acidified biochar (AB) can also effectively mitigate cadmium toxicity via ion adsorption and pH buffering. However, the combined effects of strigolactone and AB still need in-depth investigations in the context of existing literature. This study aimed to assess the individual and combined impacts of SLs (0 and 25 µM) and AB (0 and 0.75% w/w) on radish growth under Cd toxicity, i.e., 0 and 20 mg Cd/kg soil. Using a fully randomized design (CRD), each treatment was administered in four replicates. In comparison to the control under 20 mg Cd/kg soil contamination, the results showed that 25 µM strigolactone + 0.75% AB significantly improved the following: radish shoot length (~ 17%), root length (~ 47%), plant fresh weight (~ 28%), plant dry weight (~ 96%), chlorophyll a (~ 43%), chlorophyll b (~ 31%), and total chlorophyll (~ 37%). It was also noted that 0.75% AB was more pronounced in decreasing antioxidant activities than 25 µM strigolactone under 20 mg Cd/ kg soil toxicity. However, performing 25 µM strigolactone + 0.75% AB was far better than the sole application of 25 µM strigolactone and 0.75% AB in decreasing antioxidant activities in radish plants. In conclusion, by regulating antioxidant activities, 25 µM strigolactone + 0.75% AB can increase radish growth in cadmium-contaminated soils.


Acidified biochar
The leaves waste was collected from local mango orchard.After being sun-dried, the obtained waste was pyrolyzed at 470 ± 8 °C in an aerobic environment.After the pyrolysis process, the material was left to cool down before being crushed and ground to produce particles smaller than 2 mm in size.To make acidified biochar, 60 ml concentrated sulfuric acid was mixed in 1 kg of biochar.Subsequently, the acidified biochar (AB) was again dried in sun and then appropriately stored in plastic jars.The properties of biochar are provided in Table 1.

Pot filling and sowing
Acidified biochar (0 and 0.75% w/w) was mixed manually in soil, and then the mixture of AB and soil was filled into plastic bags (20 cm width and 45 cm in depth).In every plastic bag 10 kg soil was filled as per treatment plan.Each pot initially had five radish seeds sown, later 2 seedlings were retained by thinning following germination..

Antioxidants
To evaluate SOD activity, we utilized nitro blue tetrazolium (NBT), and the absorbance was measured at 560 nm to determine the final reading 26 .POD activity was assessed by observing the oxidation of guaiacol, with the absorbance recorded at 470 nm for analysis 27 .CAT activity was assessed by observing the degradation of hydrogen peroxide, which led to a decrease in absorbance at 240 nm for analysis 28 .We noticed the oxidation of ascorbate in the presence of H 2 O 2 , observing a decrease in absorbance at 290 nm 29 .The study measured the level of MDA, a dependable indicator of lipid peroxidation, by reacting the sample extract with thiobarbituric acid, leading to the formation of a colored complex, and then measuring its absorbance at 532 nm.Glutathione reductase activity was evaluated by assessing the rate of NADPH oxidation, with the decrease in absorbance at 340 nm observed for analysis 30 .Glutathione (GSH) was analyzed by adding 5% sulfosalicylic acid to the homogenate, followed by centrifugation for 10 min at 12,000×g.The resulting supernatant was then combined with 100 mM phosphate buffer and 5,5′-dithiobis(2-nitrobenzoic acid) before being measured spectrophotometrically at 412 nm 31 .To assess ascorbate (AsA) levels, an equal volume of 10% trichloroacetic acid was added to the homogenate, followed by centrifugation for 10 min at 12,000×g.The supernatant obtained was used for the spectrophotometric determination of AsA at 525 nm, following the described method 32 .

Electrolyte leakage
Electrolyte leakage was performed by dipping 1 cm diameter leaf sections in test tubes containing 20 ml of deionized water.The tubes were incubated at 25 °C for 24 h, then measured for electrical conductivity (EC1).After a 20-min heat treatment in a 120 °C water bath, the 2nd electrical conductivity (EC2) was recorded 33 .

Free proline
Free proline was quantified using a method outlined by 34 , followed by extraction using sulfosalicylic acid, glacial acetic acid and ninhydrin solutions, heating at 100 °C, and adding 5 ml of toluene.The absorbance of the toluene layer was measured at 520 nm.

Statistical analysis
The data was subjected to conventional statistical analysis 35 .OriginPro software was used to do a two-way ANOVA.OriginPro was used to carry out paired comparisons, convex hull, and hierarchical cluster analysis 36 .

Ethics approval and consent to participate
We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue.So, it is not applicable.We confirmed that all methods were performed in accordance with the relevant guidelines/regulations/legislation.

RWC
An enhancement of ~ 5, ~ 12 and ~ 20% in plant fresh weight was noted in 25 µM SLs, 0.75% AB and 25 µM SLs + 0.75% AB than control respectively.In case of 20Cd stress, SLs resulted in a ~ 5% while 25 µM SLs + 0.75% AB caused ~ 28% improvement in plant fresh weight over control.On the other hand, AB treatment showed ~ 17% improvement in plant fresh weight compared to control under 20Cd stress (Fig. 1B).
In no Cd stress, the addition of 25 µM SLs resulted in ~ 8% improvement in the plant dry weight than control.When 25 µM SLs + 0.75% AB and 0.75% AB were applied, the plant dry weight exhibited a ~ 28 and ~ 17% enhancement over control under no Cd stress.In the presence of 20Cd stress, SLs treatment caused ~ 35%, 25 µM SLs + 0.75% AB ~ 96% and 0.75% AB ~ 57% rise in plant dry weigh over the control (Fig. 1B).

Convex hull and hierarchical cluster analysis
Data points are marked with their respective treatment groups: Control, 25 µM SLs, 0.75AB, and 25 µM SLs + AB.The Control group showed a tight cluster characterized by data points with predominantly negative scores on both PC 1 and PC 2. Conversely, the 25 µM SLs treatment group forms a marked cluster, with data points showing predominantly positive scores on both PC 1 and PC 2, indicative of a treatment-specific response.Positioned between the Control and 25 µM SLs groups, the 0.75AB treatment group occupies a distinct section, suggesting a response that deviates both treatments.Notably, the 25 µM SLs + AB treatment group forms a unique cluster, positioned prominently in the positive region of PC 1 and PC 2, underscoring its distinctive response to the combined treatment (Fig. 6A).
In this analysis, two main clusters are identified, which correspond to different heavy metal (HM) treatments.The first cluster, labeled no Cd, encompasses data points characterized by positive values along both PC 1 and PC 2 axes.These data points exhibit increasing values along both principal components.The convex hull surrounding these points describes the boundary of this cluster, indicating the range of variation within this group.The second cluster, labeled 20Cd, includes data points characterized by negative values along both PC 1 and PC 2 axes.These data points exhibit decreasing values along both principal components (Fig. 6B).
Chlorophyll a and total chlorophyll are tightly associated, evidenced by their similarity score of 0.30045, indicating a close relationship between these two variables.RWC is clustered with a high similarity score of 0.50822, indicating a strong link to physiological parameters concerning water content.Plant fresh weight and plant dry weight exhibit a strong relationship, clustered tightly with a similarity score of 0.51475, representing plant biomass.Shoot length and carotenoids also demonstrate a notable similarity score of 0.52585, indicating shared patterns within the dataset.Chlorophyll b clusters with SOD and POD, suggesting a potential correlation between chlorophyll content and enzymatic antioxidant activities.H 2 O 2 and MDA form a cluster with a high similarity score of 1.13996, indicating a possible association between these oxidative stress markers.CAT is grouped with root length, indicating a potential relationship between catalase activity and root development.Protein content stands separately with a similarity score of 1.321, suggesting distinct patterns compared to other variables.Leaves Cd and root Cd exhibit a high similarity score of 1.58953, suggesting a close association between cadmium concentrations in leaves and roots.Electrolyte leakage and APX are grouped with a similarity score of 1.76274, indicating at a potential link between electrolyte leakage and ascorbate peroxidase activity.Moreover, clusters with very high similarity scores, including one exceptionally high score (97.71591), denote distinct patterns or relationships within those clusters (Fig. 6C).

Pearson correlation analysis
Shoot length exhibited a perfect positive correlation with itself, as expected.Root length showed a very strong positive correlation with shoot length (r = 0.97577), indicating that as shoot length increases, root length tends to increase proportionally.Moreover, plant fresh weight plant dry weight exhibited very strong positive www.nature.com/scientificreports/correlations with both shoot and root lengths, indicating a strong association between plant size and biomass (r = 0.97994-0.99111).Chlorophyll (a.b, and total) and carotenoids all showed very strong positive correlations with each other and with plant biomass measures (r = 0.96005-0.99481).Conversely, electrolyte leakage, a measure of cellular membrane integrity, showed strong negative correlations with plant size, chlorophyll content, and biomass (r = − 0.967 to − 0.98827).Enzymatic antioxidant activities, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), demonstrated negative correlations with most growth and physiological parameters, indicating their involvement in response to stress conditions (r = − 0.50606 to − 0.38464).Hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA), markers of oxidative stress, also exhibited strong negative correlations with most parameters, indicating their association with plant health and stress response (r = − 0.98106 to − 0.97233).Additionally, heavy metal accumulation in leaves and roots, represented by leaves Cd and root Cd, displayed strong negative correlations with various growth and physiological parameters, suggesting their detrimental effects on plant growth and health (r = − 0.95299 to − 0.93952) (Fig. 7).

Discussion
In current study a significant decline in root length, shoot length, plant fresh weight, plant dry weight was chlorophyll contents observed in radish plants which were cultivated in 20 mg Cd/kg soil toxicity.This decline was associated with oxidative stress induced by Cd contamination 37 .Cadmium primarily accumulates in plant roots.
High concentrations of Cd in the soil can inhibit root growth directly by damaging root cells and impairing root development 38 .This inhibition can occur due to the disruption of cell division, elongation, and differentiation processes in the root tips.As a result, the overall root length decreases 39 .One of the reasons for reduced shoot length is the impairment of water and nutrient uptake due to damaged roots.Cadmium can interfere with the uptake of essential nutrients such as iron, magnesium, and calcium, which are crucial for plant growth and development 40 .Moreover, Cd can also disrupt photosynthesis by affecting chlorophyll synthesis and photosystem functioning, leading to reduced biomass production and consequently, shorter shoots 41 .Cadmium interferes with various metabolic processes, including photosynthesis, respiration, and protein synthesis, leading to decreased plant productivity and biomass accumulation 42 .Additionally, Cd can induce oxidative stress by generating reactive oxygen species (ROS) and disrupting antioxidant defense mechanisms.This oxidative stress damages cellular structures and biomolecules, further contributing to reduced biomass production and weight 43,44 .
SLs have been implicated in regulating root system architecture, particularly in response to nutrient availability and environmental stresses 13 .By modulating root architecture, SLs can potentially enhance root growth and function even in the presence of Cd toxicity.It can promote the formation of lateral roots and increase root hair density, which may improve nutrient and water uptake efficiency and mitigate the negative impact of Cd on root growth.Additionally, the application of SLs can enhance photosynthesis by stimulating the development of chloroplasts, which in turn results in an increase in chlorophyll content 45 .This hormone is believed to likely stimulate the expression of genes responsible for chlorophyll biosynthesis and maintenance.and play a crucial role in shielding against photodamage 46 .The study found that the combination of biochar and SLs enhanced antioxidant activities under Cd stress, including POD, SOD, CAT, and APX.These enzymes play a crucial role in reducing reactive oxygen species (ROS) levels in plants.CAT decomposes hydrogen peroxide into water and oxygen, while SOD converts superoxide radicals into H2O2 and oxygen.POD and APX both function in the detoxification of H 2 O 2 .These enzymes collectively contribute to the breakdown of ROS, preventing oxidative damage to cellular components such as lipids, proteins, and DNA 47 .Biochar has a high surface area and contains functional groups that can adsorb and immobilize Cd ions 2,5 .By binding to the surface of biochar, Cd becomes less mobile in the soil and is less likely to be taken up by plant roots.It may compete with plant roots for Cd uptake by adsorbing Cd ions onto its surface.This competitive sorption reduces the concentration of Cd ions available for plant uptake, further decreasing Cd accumulation in plant tissues 48 .Furthermore, acidified biochar can improve soil structure, water retention, and nutrient availability, which indirectly affects plant growth and Cd uptake 49 .Healthier plants grown in well-structured soils may exhibit reduced Cd uptake due to improved physiological conditions.It also serves as a habitat and substrate for soil microorganisms.Microbes associated with biochar can contribute to the biodegradation of organic contaminants and the immobilization of heavy metals through various biological processes.Enhanced microbial activity can contribute to the transformation and sequestration of Cd in the soil, reducing its availability to plants [50][51][52] .

Conclusion
In conclusion, the combined application of 0.75% AB and SLs (25 µM) demonstrates promising efficacy in mitigating soil cadmium (Cd) toxicity and enhancing radish cultivation when compared to their individual application and control.The observed reduction in the levels of antioxidants SOD, POD, and CAT under cadmium toxicity (20 mg/kg) with the application of AB and SLs suggests a lower uptake of cadmium in plants.Consequently, the application of AB and SLs in cadmium-contaminated soils hold potential for mitigating cadmium toxicity and achieving higher radish growth.

Figure 1 .
Figure 1.The impact of SLs and biochar on radish shoot and root length (A), plant fresh and dry weight (B) grown in no Cd and 20Cd stress.The graph shows the average of 4 replicates with±, where significant differences (p ≤ 0.05) are indicated by distinct letters on bars, determined by the Tukey test.

Figure 2 .
Figure 2. The influence of SLs and biochar on the radish chlorophyll a, b, total and carotenoids grown in no Cd 20Cd stress.graph shows the average of 4 replicates with±, where significant differences (p ≤ 0.05) are indicated by distinct letters on bars, determined by the Tukey test.

Figure 3 .
Figure 3.The impact of SLs and biochar on radish protein content, electrolyte leakage, and relative water content grown in no Cd and 20Cd stress.The graph shows the average of 4 replicates with ± , where significant differences (p ≤ 0.05) are indicated by distinct letters on bars, determined by the Tukey test.

Figure 4 .
Figure 4.The impact of SLs and biochar on radish H 2 O 2 , malondialdehyde, leaf and root Cd concentration grown in no Cd and 20Cd stress.The graph shows the average of 4 replicates with±, where significant differences (p ≤ 0.05) are indicated by distinct letters on bars, determined by the Tukey test.

Figure 5 .
Figure 5.The impact of SLs and biochar on superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) levels in radish grown in no Cd stress and 20Cd stress.The graph shows the average of 4 replicates with±, where significant differences (p ≤ 0.05) are indicated by distinct letters on bars, determined by the Tukey test.

Figure 6 .
Figure 6.Cluster plot convex hull for treatments (A), Cd levels (B), and hierarchical cluster plot (C) for studied attributes.